Method of identifying hairpin DNA probes by partial fold analysis

ABSTRACT

Method of identifying molecular beacons in which a secondary structure prediction algorithm is employed to identify oligonucleotide sequences within a target gene having the requisite hairpin structure. Isolated oligonucleotides, molecular beacons prepared from those oligonucleotides, and their use are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/533,894, filed Jan. 2, 2004, which is herebyincorporated by reference in its entirety.

This invention was made, at least in part, with funding received fromthe U.S. Department of Energy under grant DE-FG02-02ER63410.000. TheU.S. government may retain certain rights in this invention.

FIELD OF THE INVENTION

The present invention generally relates to the use of DNA hairpins asmolecular beacon probes. More specifically, the present invention isdirected to methods of generating highly specific and highly selectivemolecular beacon probes by using naturally occurring DNA hairpinspresent in organisms of interest.

BACKGROUND OF THE INVENTION

Methods for the rapid detection and serotyping of pathogens are of highinterest, due in part to the dramatic improvement in treatment efficacyfor a bacterial or viral infection diagnosed early relative to onediagnosed at a later stage (Inglesby et al., “Anthrax as a BiologicalWeapon: Medical and Public Health Management,” J. Am. Med. Assoc.281:1735-1745 (1999)). Unfortunately, most current methods of pathogenidentification rely on some level of sample manipulation, (enrichment,fluorescent tagging, etc.) which can be costly in terms of both time andmoney. Thus, eliminating sample labeling will result in a significantsavings and has the potential to speed diagnosis. The use of DNAhairpins as “molecular beacons” (Broude, “Stem-loop Oligonucleotides: aRobust Tool for Molecular Biology and Biotechnology,” Trends Biotechnol.20:249-256 (2002)), either in solution (Tyagi et al., “MolecularBeacons: Probes that Fluoresce upon Hybridization,” Nature Biotech.19:365-370 (2001); Dubertret et al., “Single-mismatch Detection UsingGold-quenched Fluorescent Oligonucleotides,” Nature Biotech. 19:365-370(2001)) or immobilized on a solid surface (Fang et al., “Designing aNovel Molecular Bacon for Surface-Immobilized DNA HybridizationStudies,” J. Am. Chem. Soc. 121:2921-2922 (1999); Wang et al., “LabelFree Hybridization Detection of Single Nucleotide Mismatch byImmobilization of Molecular Beacons on Agorose Film,” Nucl. Acids. Res.30:61 (2002); Du et al., “Hybridization-based Unquenching of DNAHairpins on Au Surfaces: Prototypical “Molecular Beacon” Biosensors,” J.Am. Chem. Soc. 125:4012:4013 (2003); Fan et al., “ElectrochemicalInterrogation of Conformational Changes as a Reagentless Method for theSequence-specific Detection of DNA,” Proc. Natl. Acad. Sci. USA100:9134-9137 (2003)), has proven to be a useful method for “label-free”detection of oligonucleotides. Molecular beacons consist of DNA hairpinsfunctionalized at one terminus with a fluorophore and at the otherterminus with a quencher. In the absence of their complement, they existin a closed, “dark” conformation. Hybridization occurs on introductionof complementary oligonucleotides, which concomitantly forces open thehairpin and allows for a fluorescent, “bright” state.

Traditionally, as illustrated in FIG. 1, molecular beacons have beendesigned by supplementing the targeted DNA sequence at both termini withadditional self-complementary nucleotides to force the formation of ahairpin (Monre et al., “Molecular Beacon Sequence Design Algorithm,”Biotechniques 34:68-73 (2003)). While generally successful, the additionof non target-derived nucleotides increases the potential fornon-specific binding, thus potentially reducing both the sensitivity andselectivity of the probe beacon. Modifications of this discoveryprotocol, such as the “shared stem” methodology of Bao and coworkers(Tsourkas et al., “Structure-function Relationships of Shared-Stem andConventional Molecular Beacons,” Nucl. Acids Res. 30:4208-4215 (2002)),still incorporate several bases unrelated to the target sequence. Thus,the latter approach potentially suffers from the same deficiencies. Itwould be desirable to identify a reliable approach for identifying DNAhairpins that overcomes the above-noted deficiencies.

The present invention is directed to achieving these objectives andotherwise overcoming the above-noted deficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a method ofidentifying hairpin nucleic acid probes. The method includes the stepsof: providing a target nucleic acid sequence that is larger than about100 nucleotides in length; predicting a folded structure of the targetnucleic acid sequence; identifying the nucleotide sequence of a hairpinwithin the folded structure of the target nucleic acid sequence; andpredicting a folded structure of the identified nucleotide sequence ofthe hairpin, in the absence of other nucleotides of the target nucleicacid sequence, wherein the folded structure of the hairpin has apredicted E value of at most about −3 kcal/mol.

A second aspect of the present invention relates to a method ofpreparing a molecular beacon. The method includes the steps of:providing a hairpin nucleic acid probe identified according to the firstaspect of the present invention; and tethering a fluorescent label and aquenching agent to the opposed termini of the provided hairpin nucleicacid probe to form a molecular beacon, wherein the molecular beacon issubstantially non-fluorescent in the absence of a nucleic acidcomplementary to the hairpin nucleic acid probe.

A third aspect of the present invention relates to a method of preparinga hairpin nucleic acid molecule. This method includes the steps ofidentifying the nucleotide sequence of a hairpin in accordance with thefirst aspect of the present invention; and synthesizing the identifiedhairpin nucleic acid molecule.

A fourth aspect of the present invention relates to an isolated nucleicacid molecule prepared according to the third aspect of the presentinvention.

A fifth aspect of the present invention relates to an isolated molecularbeacon that includes a nucleic acid molecule according to the fourthaspect of the present invention; a fluorescent label tethered to oneterminus of the nucleic acid molecule; and a quenching agent tethered tothe other terminus of the nucleic acid molecule.

Additional aspects of the present invention relate to the use of thehairpin nucleic acid molecules and molecular beacons as probes in thedetection of target nucleic acid molecules, according to any of avariety of hybridization-based detection procedures.

The ability to rapidly detect the presence of biological agents in theenvironment is of keen interest to the civilian and military healthcommunities. The use of DNA hairpins as “molecular beacons” has proven auseful method for the detection of bacterial oligonucleotides. Thepresent invention affords a significant improvement over previouslyemployed molecular beacons by using naturally occurring DNA hairpins asmolecular beacon probes. This circumvents the need for supplementationwith additional bases; as noted in the Examples, supplementation ormodification of the naturally occurring hairpins is likely to result inenergetically less favorable complementation.

The working examples of the present invention demonstrate thesignificant specificity and energetically stable target/hairpindimerizations, thus producing viable molecular beacons for varyingexperimental conditions, probes and fluorophores. By selecting probesbased on their predicted structures and free energy, and by controllingprobe length, the present invention affords a systematic approach forpreparing nucleic acid probes and molecular beacons that can be used toselectively and sensitively discriminate between target and non-targetmolecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the prior art method of DNA hairpin probe designdemonstrating the section of non-pairing sequences present in the finalcomplex.

FIG. 2 shows the final probe/target complex of the present inventionwhen the probe is selected based on total sequence complementarity.

FIG. 3 shows the predicted secondary structure and hairpin regionsselected from Bacillus anthracis. A partial gene sequence of theBacillus anthracis pag gene (isolate IT-Carb3-6254) (Adone et al., J.Appl. Microbio. 92:1-5 (2002), which is hereby incorporated by referencein its entirety) was obtained from GenBank Accession AJ413936, which ishereby incorporated by reference in its entirety. The secondarystructure of ˜1000 nucleotide fragment (SEQ ID NO: 1) of theaforementioned sequence was then computationally predicted usingRNAstructure v. 3.7 (Mathews et al., J. Mol. Biol. 288:911-940 (1999),which is hereby incorporated by reference in its entirety).

FIGS. 4A-B show structural predictions for two excised sequences:BaPag668-706 (Pag668-706) (SEQ ID NO: 2) andBaPag1208-1241(Pag1208-1241) (SEQ ID NO: 3). The sequences are isolatedfrom the full sequence and subjected to secondary structure predictions.The number of nucleotides is indicated by nt count.

FIG. 5 demonstrates that the specificity of the BaPag 668-706 hairpinfor its target is supported by a BLAST search of the GenBank databaseusing the BaPag 668-706 sequence. A clear demarcation exists betweentarget scores (of 78) and non-target scores (of 42 and lower) indicatingthat only sequences from Bacillus anthracis, the target organism, havehigh scores; whereas other “matching sequences” from non-targetorganisms have significantly lower scores.

FIG. 6 shows the predicted secondary structure and hairpin regionsselected from the Staphylococcus aureus genome, a segment of which (SEQID NO: 4) was obtained from Genbank Accession AP003131, which is herebyincorporated by reference in its entirety. The secondary structure ofthe obtained segment was predicted using computer program RNAStructureversion 3.7 (Mathews et al., J. Mol. Biol. 288:911-940 (1999), which ishereby incorporated by reference in its entirety). From this predictedstructure, two naturally occurring hairpins were identified, onecorresponding to AH2 and the other corresponding to BH2.

FIGS. 7A-B show structural predictions for two excised sequences: AH2(SEQ ID NO: 5) and BH2 (SEQ ID NO: 6). The sequences were isolated fromthe full sequence and subjected to second structure predictions. The AH2sequence appears primarily to target an intergenic region betweenORFID:SA0529 and ORFID:SA0530, and the BH2 sequence appears to target anintergenic region between ORFID:SA0529 and ORFID:SA0530 but alsoincludes several bases within the latter open reading frame.

FIGS. 8A-D show the final structural prediction of BaPag 668-706 (SEQ IDNO: 2), BaPag 1208-1241 (SEQ ID NO: 3), AH2 (SEQ ID NO: 5), and BH2 (SEQID NO: 6) in duplex with their corresponding complements (SEQ ID NOS:7-10, respectively). Having confirmed that the selected hairpin(s)satisfy initial selected criteria, a final structural prediction of thesequence in duplex with its complement was computed. Each of theseduplexes have a predicted E value that is about nine to ten-fold greaterthan the predicted E value (or ΔΔG value) for the hairpin alone, andtherefore they are expected to favorably form a duplex with theirtargets.

FIG. 9 demonstrates that hairpins favorably hybridize with their targetDNA. Samples of BaPag668 (BaPag668-706) and BaPag1208 (BaPag1208-1241),both alone and mixed with equal amount of complement, were run on anative polyacrylamide gel. The presence of single bands in Lanes 1 and 3is evidence that the hairpins preferentially adopt one structure becauseany variations from the predicted structure would either enhance orretard the variant's migration through the gel, thus creating multiplebands. The upward shift seen in Lanes 2 and 4 is indicative of theaddition of mass that occurs during the hybridization of the hairpinswith their targets. The increased contrast of the bands in Lanes 2 and 4also gives indication that the hairpins are successfully formingdouble-stranded duplexes with their targets, as the dye usedpreferentially binds double-stranded regions of DNA.

FIGS. 10A-H show the thermal melting curves for DNA hairpin probes.Unmodified versions of BaPag668-706, BaPag1208-1241, AH2, BH2, and theircomplements were purchased from Invitrogen (cartridge purity). Allthermal melts were conducted on a Gilford spectrophotometer, with theoligonucleotides dissolved in 0.5 M NaCl Buffer (20 mM cacodylic acid,0.5 mM EDTA, and 0.5 M NaCl, pH=7.28). Samples were warmed to 90° C. andsubsequently cooled to 10° C. prior to running melts. Solutiontemperatures were raised by 1° C. per minute over a range of 15° C. to90° C. and data points were collected approximately every 30 s (FIGS.10A-D). All melting temperatures (x-axis) of BaPag668-706,BaPag1208-1241, AH2, and BH2 were found to be concentration independent(absorbance is indicated on the y-axis). The unmodified hairpins werethen mixed with a ten-fold excess of complementary DNA and a secondseries of melting profiles were obtained (FIGS. 10E-H). As was expected,introduction of complement to the hairpins produced a biphasictransition curve, with the first transition corresponding to thelinearization of the target DNA, which is also believed to possessordered secondary structure, and the second, higher temperature,transition corresponding to the melting point of the duplex DNA.

FIG. 11 shows the solution phase performance of the BaPag668-706 probe.BaPag668-706 was purchased from Integrated DNA Technologies, Inc. as amolecular beacon using 5′-fluorescein and 3′-dabcyl as the fluorophoreand quencher, respectively. BaPag668-706 was diluted to a concentrationof 300 nM in 0.5 M NaCl Buffer (20 mM Cacodylic acid, 0.5 mM EDTA, and0.5 M NaCl, pH=7.28), to which target DNA was then added such that thefinal ratio of target to beacon ranged from 1:1 to 4:1. Samples wereallowed to incubate 5 hours at room temperature and were kept out ofdirect light as much as possible prior to excitation to preventphotobleaching. Samples were transferred to a Starna Cells 23-Q-10Quartz fluorometer cell (10 mm pathlength) and placed on an ActonResearch Instruments Fluorometer System. The fluorophore was excited at490 nm and the resulting emission was monitored from 500 to 620 nm(x-axis). BaPag668-706 exhibits minimal fluorescence alone, and, asexpected, addition of the target complementary oligonucleotide causesfluorescence to increase in a concentration-dependent manner.

FIGS. 12A-F show the performance of the BaPag1208-1241 probe immobilizedin a 1:10 ratio with mercaptopropanol on an Au-film. The 5′-thiolterminated version of BaPag1208-1241 was immobilized on a thin Au filmin the presence of mercaptopropanol as described previously (Du et al.,“Hybridization-based Unquenching of DNA Hairpins on Au Surfaces:Prototypical “Molecular Beacon” Biosensors,” J. Am. Chem. Soc.,125:4012:4013 (2003), which is hereby incorporated by reference in itsentirety) with the only major change being the use of 0.5 M NaCl bufferas the diluent as opposed to deionized water. When immobilized on anAu-film in a 1:10 ratio with mercaptopropanol, BaPag1208-1241 showsgreater than an 18-fold increase in fluorescence intensity (y-axis) inresponse to incubation in a 2.5 μM target solution (FIGS. 12A-C). Whenthe concentration of the target solution is lowered to 1.0 μM, theobserved response drops to about 10-fold, which is still significant(FIGS. 12D-F).

FIGS. 13A-F show the performance of the BaPag1208-1241 probe immobilizedin a 1:1 ratio with mercaptopropanol on an Au-film. BaPag1208-1241 wasimmobilized onto an Au-film with mercaptopropanol in a 1:1 ratio andsubjected to the same target concentrations as described previously. Asseen in FIGS. 13A-C, when immobilized in a 1:1 ratio withmercaptopropanol, BaPag1208 shows a superior response, as measured byfluorescence intensity (y-axis), to target over that observed when theimmobilization ratio is 1:10. This increased response is especiallysignificant at lower concentrations as is evidenced by the greater than20-fold intensity increase observed after incubation in 1.0 μM target(FIGS. 13D-F).

FIG. 14 summarizes the sensitivity of BaPag1208-1241 to a targetsequence when immobilized onto an Au-film. The nanomolar targetconcentration is depicted on the x-axis. The fold increase in binding ofBaPag1208-1241 to a target sequence is depicted on the y-axis.

FIGS. 15A-F show the use of Au-immobilized AH2 and BH2 beacons to detectcomplementary DNA sequences. The AH2 and BH2 hairpins were 3′-modifiedwith tetramethylrhodamine (“TAMRA”) and Cy5, respectively. Each probewas dissolved separately in a solution of mercaptopropanol and water(1:10 molar ratio of probe and mercaptopropanol). The resulting probesolutions were then mixed in a 1:1 ratio, added to Au-chips andmeasurements of baseline fluorescence made (FIGS. 15B and 15E). TheAu-films were then separately incubated in their appropriatecomplementary target solutions and fluorescence measured (FIGS. 15C and15F). The fluorescence demonstrated in FIGS. 15C and 15F indicate thatAH2 and BH2 probe beacons effectively detect complementary DNAsequences.

FIGS. 16A-C show the calculated hybridization energies forfolding-derived and modified BaPag668-706 beacons. FIG. 16A shows thesame secondary structure as in FIG. 4A (SEQ ID NO: 2). The termini ofprobe BaPag668-706 was extended by the self-complementary sequence[d(CGACG)]₂ (SEQ ID NO: 11), then the hybridization energy calculated(FIG. 16B). Five bases were removed from each end of BaPag668-706,replaced with [d(CGACG)]₂, and the hybridization energy again calculated(FIG. 16C). BaPag673 corresponds to BaPag668 with 5 bases removed fromeach end (SEQ ID NO: 12). The complementary sequence of BaPag668-706 isalso shown (SEQ ID NO: 7). In each case, calculated ΔΔG was lessfavorable for the modified beacons than for the probes derived directlyfrom folding.

FIGS. 17A-C show the calculated hybridization energies forfolding-derived and modified BaPag1208-1241 beacons. FIG. 17A shows thesame secondary structure as in FIG. 4B (SEQ ID NO: 3). The termini ofprobe BaPag1208-1241 was extended by the self-complementary sequence[d(CGACG)]₂ (SEQ ID NO: 13), then the hybridization energy calculated(FIG. 17B). The complementary sequence of BaPag1208-1241 is also shown(SEQ ID NO: 8). Five bases were removed from each end of BaPag1208-1241,replaced with [d(CGACG)]₂ (SEQ ID NO: 14), and the hybridization energyagain calculated (FIG. 17C). BaPag1213 corresponds to BaPag1208 with 5bases removed from each end. In each case, calculated ΔΔG was lessfavorable for the modified beacons than for the probes derived directlyfrom folding.

DETAILED DESCRIPTION OF THE INVENTION

The method of the invention involves obtaining or providing a probenucleotide sequence from a molecular target. The target nucleotidesequence can be sequenced from an isolated cDNA or obtained from anonline database such as GenBank. Regardless of the source of the targetnucleotide sequence, a partial fold analysis is performed on thenucleotide sequence using any of a variety of suitable folding softwaresuch as, e.g., RNAStructure program (available from D. Turner at theUniversity of Rochester, Rochester, N.Y.), Mfold software package(available from M. Zucker at the Rensselear Polytechnic Institute,Rensselear, N.Y.), and Vienna RNA software package, including RNAfold,RNAeval, and RNAsubopt (available from I. Hofacker at the Institute forTheoretical Chemistry, Vienna, Austria). With respect to theRNAStructure program, applicants have discovered that segments largerthan approximately 1000 bases would crash the program RNAstructure v.3.7. Thus, it may or may not be possible to predict the secondarystructure of an entire nucleic acid molecule depending on the lengththereof. Ideally, the secondary structure of the entire sequence wouldbe predicted, but as demonstrated in the examples that is not necessary.

The resulting folded structure may or may not be the true activeconformation of the RNA molecule in a cellular environment; however, itrepresents the lowest free energy state as predicted using suchsoftware. It is believed that more often than not, the predicted lowestfree energy state of the nucleic acid molecule sufficiently resemblesthe true active conformation. Nonetheless, the resulting foldedstructure is analyzed to identify hairpin regions thereof.

Having identified hairpin structures within the folded structure of theprospective target nucleic acid molecule, the hairpin sequences areisolated from the larger sequence (i.e., that was used as input to thefolding software). The isolation can be performed in silico. Onceisolated, the hairpin sequence is subjected to a second structuralprediction as was performed on the prospective target nucleic acidmolecule.

The overall length of the selected hairpin is preferably between about12 and about 60 nucleotides, more preferably between about 20 and about50 nucleotides, most preferably between about 30 and about 40nucleotides. It should be appreciated, however, that longer or shorternucleic acids can certainly be used. According to the preferredhairpins, the regions forming the stem of the hairpin are preferably atleast about 4 nucleotides in length and up to about 28 nucleotides inlength, depending on the overall length of the nucleic acid probe andthe size of a loop region present between the portions forming the stem.It is believed that a loop region of at least about 4 or 5 nucleotidesis needed to form a stable hairpin. The regions forming the stem can beperfectly matched (i.e., having 100 percent complementary sequences thatform a perfect stem structure of the hairpin conformation) or less thanperfectly matched (i.e., having non-complementary portions that formbulges within a non-perfect stem structure of the hairpin conformation).When the first and second regions are not perfectly matched, the regionsforming the stem structure can be the same length or they can bedifferent in length.

Importantly, applicants have found that the predicted E value for thehairpin should preferably be at most about −3 kcal/mol, more preferablyat most about −3.5 kcal/mol, most preferably between about −4 kcal/moland about −12 kcal/mol. It should be appreciated, however, thatidentified hairpins can still function as molecular probes if theirpredicted E value falls outside these ranges.

Once the structure of the hairpin itself has been predicted, the duplexformed between the hairpin and its complement is subjected to astructural prediction as was performed on the prospective target nucleicacid molecule and the hairpin. This step, not necessary foridentification of the hairpin per se, is performed primarily to ensurethat the hybridization of the two sequences (hairpin and complement),and thus the disruption of the hairpin, will be an energeticallyfavorable process. Ideally there should be an increase in the predictedE value preferably at least about a two-fold increase, more preferablyat least about a five-fold increase or even more preferably at leastabout a ten-fold increase. This structural prediction also serves todemonstrate the primary advantage of the technique: after hybridization,there are no extraneous unhybridized nucleotides and, thus, lowered riskof nonspecific binding.

To further verify the specificity of the hairpin sequence for itscomplement, the hairpin sequence can be used to perform a BLAST databasesearch (of, e.g., the GenBank database). Ideally, the resulting BLASTsearch will show not only high match scores for molecular targets (ortarget organisms), but also a sharp discrepancy (or clear demarcation)between the high match scores of the target and any match scores ofnucleic acid molecules bearing lower similarity. By sharp discrepancyand clear demarcation, it is intended that a gap of at least about 5points, preferably at least about 10 points, more preferably at leastabout 15 points, most preferably at least about 20 points, existsbetween the target and non-target sequences. This is exemplified inExample 1 below.

Having thus identified suitable hairpin nucleic acid molecules that canbe utilized for the detection of target nucleic acids and, thus, theidentification of target organisms (by virtue of hybridization betweenthe hairpin and the target), persons of skill in the art can readilysynthesize hairpin nucleic acid molecules and prepare molecular beaconscontaining the same in accordance with known procedures.

The hairpin nucleic acid molecules can be synthesized according tostandard procedures. Commercial synthesis facilities, in particular, areadept at providing this service.

Molecular beacons can be constructed by tethering to the termini of thehairpin nucleic acid molecule a fluorescent label and a quenching agent,respectively. In one embodiment, the fluorescent label is tethered tothe 5′ end of the hairpin nucleic acid molecule and the quenching agentis tethered to the 3′ end thereof. In another embodiment, thefluorescent label is tethered to the 3′ end of the hairpin nucleic acidmolecule and the quenching agent is tethered to the 5′ end thereof.

The fluorescent label can be any fluorophore that can be conjugated to anucleic acid and preferably has a photoluminescent property that can bedetected and easily identified with appropriate detection equipment.Exemplary fluorescent labels include, without limitation, fluorescentdyes, semiconductor quantum dots, lanthanide atom-containing complexes,and fluorescent proteins. The fluorophore used in the present inventionis characterized by a fluorescent emission maxima that is detectableeither visually or using optical detectors of the type known in the art.Fluorophores having fluorescent emission maxima in the visible spectrumare preferred.

Exemplary dyes include, without limitation, Cy2™, YO-PRO™-1, YOYO™-1,Calcein, FITC, FluorX™, Alexa™, Rhodamine 110, 5-FAM, Oregon Green™ 500,Oregon Green™ 488, RiboGreen™, Rhodamine Green™, Rhodamine 123,Magnesium Green™, Calcium Green™, TO-PRO™-1, TOTO®-1, JOE, BODIPY®530/550, Dil, BODIPY® TMR, BODIPY® 558/568, BODIPY® 564/570, Cy3™,Alexa™ 546, TRITC, Magnesium Orange™, Phycoerythrin R&B, RhodaminePhalloidin, Calcium Orange™, Pyronin Y, Rhodamine B, TAMRA, RhodamineRed™, Cy3.5™, ROX, Calcium Crimson™, Alexa™ 594, Texas Red®, Nile Red,YO-PRO™-3, YOYO™-3, R-phycocyanin, C-Phycocyanin, TO-PRO™-3, TOTO®-3,DiD DilC(5), Cy5™, Thiadicarbocyanine, and Cy5.5™. Other dyes now knownor hereafter developed can similarly be used as long as their excitationand emission characteristics are compatible with a light source andnon-interfering with other fluorescent labels that may be tethered todifferent hairpin nucleic acid molecules (i.e., not capable ofparticipating in fluorescence resonant energy transfer or FRET).

Attachment of dyes to the oligonucleotide probe can be carried out usingany of a variety of known techniques allowing, for example, either aterminal base or another base near the terminal base to be bound to thedye. For example, 3′-tetramethylrhodamine (TAMRA) may be attached usingcommercially available reagents, such as 3′-TAMRA-CPG, according tomanufacturer's instructions (Glen Research, Sterling, Va.). Otherexemplary procedures are described in, e.g., Dubertret et al., NatureBiotech. 19:365-370 (2001); Wang et al., J. Am. Chem. Soc.,125:3214-3215 (2003); Bioconjugate Techniques, Hermanson, ed. (AcademicPress) (1996), each of which is hereby incorporated by reference in itsentirety.

Exemplary proteins include, without limitation, both naturally occurringand modified (i.e., mutant) green fluorescent proteins (Prasher et al.,Gene 111:229-233 (1992); PCT Application WO 95/07463, each of which ishereby incorporated by reference in its entirety) from various sourcessuch as Aequorea and Renilla; both naturally occurring and modified bluefluorescent proteins (Karatani et al., Photochem. Photobiol.55(2):293-299 (1992); Lee et al., Methods Enzymol. (Biolumin.Chemilumin.) 57:226-234 (1978); Gast et al., Biochem. Biophys. Res.Commun. 80(1):14-21 (1978), each of which is hereby incorporated byreference in its entirety) from various sources such as Vibrio andPhotobacterium; and phycobiliproteins of the type derived fromcyanobacteria and eukaryotic algae (Apt et al., J. Mol. Biol. 238:79-96(1995); Glazer, Ann. Rev. Microbiol. 36:173-198 (1982); Fairchild etal., J. Biol. Chem. 269:8686-8694 (1994); Pilot et al., Proc. Natl.Acad. Sci. USA 81:6983-6987 (1984); Lui et al., Plant Physiol.103:293-294 (1993); Hournard et al., J. Bacteriol. 170:5512-5521 (1988),each of which is hereby incorporated by reference in its entirety),several of which are commercially available from ProZyme, Inc. (SanLeandro, Calif.). Other fluorescent proteins now known or hereafterdeveloped can similarly be used as long as their excitation and emissioncharacteristics are compatible with the light source and non-interferingwith other fluorescent labels that may be present.

Attachment of fluorescent proteins to the oligonucleotide probe can becarried out using substantially the same procedures used for tetheringdyes to the nucleic acids, see, e.g., Bioconjugate Techniques,Hermanson, ed. (Academic Press) (1996), which is hereby incorporated byreference in its entirety.

Nanocrystal particles or semiconductor nanocrystals (also known asQuantum Dot™ particles), whose radii are smaller than the bulk excitonBohr radius, constitute a class of materials intermediate betweenmolecular and bulk forms of matter. Quantum confinement of both theelectron and hole in all three dimensions leads to an increase in theeffective band gap of the material with decreasing crystallite size.Consequently, both the optical absorption and emission of semiconductornanocrystals shift to the blue (higher energies) as the size of thenanocrystals gets smaller. When capped nanocrystal particles of theinvention are illuminated with a primary light source, a secondaryemission of light occurs at a frequency that corresponds to the band gapof the semiconductor material used in the nanocrystal particles. Theband gap is a function of the size of the nanocrystal particle. As aresult of the narrow size distribution of the capped nanocrystalparticles, the illuminated nanocrystal particles emit light of a narrowspectral range resulting in high purity light. Particles size can bebetween about 1 nm and about 1000 nm in diameter, preferably betweenabout 2 nm and about 50 nm, more preferably about 5 nm to about 20 nm.

Fluorescent emissions of the resulting nanocrystal particles can becontrolled based on the selection of materials and controlling the sizedistribution of the particles. For example, ZnSe and ZnS particlesexhibit fluorescent emission in the blue or ultraviolet range (˜400 nmor less); Au, Ag, CdSe, CdS, and CdTe exhibit fluorescent emission inthe visible spectrum (between about 440 and about 700 nm); InAs and GaAsexhibit fluorescent emission in the near infrared range (˜1000 nm), andPbS, PbSe, and PbTe exhibit fluorescent emission in the near infraredrange (i.e., between about 700-2500 nm). By controlling growth of thenanocrystal particles it is possible to produce particles that willfluoresce at desired wavelengths. As noted above, smaller particles willafford a shift to the blue (higher energies) as compared to largerparticles of the same material(s).

Preparation of the nanocrystal particles can be carried out according toknown procedures, e.g., Murray et al., MRS Bulletin 26(12):985-991(2001); Murray et al., IBM J. Res. Dev. 45(1):47-56 (2001); Sun et al.,J. Appl. Phys. 85(8, Pt. 2A): 4325-4330 (1999); Peng et al., J. Am.Chem. Soc. 124(13):3343-3353 (2002); Peng et al., J. Am. Chem. Soc.124(9):2049-2055 (2002); Qu et al., Nano Lett. 1(6):333-337 (2001); Penget al., Nature 404(6773):59-61 (2000); Talapin et al., J. Am. Chem. Soc.124(20):5782-5790 (2002); Shevenko et al., Advanced Materials14(4):287-290 (2002); Talapin et al., Colloids and Surfaces, A:Physiochemical and Engineering Aspects 202(2-3):145-154 (2002); Talapinet al., Nano Lett. 1(4):207-211 (2001), each of which is herebyincorporated by reference in its entirety. Alternatively, nanocrystalparticles can be purchased from commercial sources, such as EvidentTechnologies.

Attachment of a nanocrystal particle to the oligonucleotide probe can becarried out using substantially the same procedures used for tetheringdyes thereto. Details on these procedures are described in, e.g.,Bioconjugate Techniques, Hermanson, ed. (Academic Press) (1996), whichis hereby incorporated by reference in its entirety.

Exemplary lanthanide atoms include, without limitation, Ce, Pr, Nd, Pm,Sm, Eu, Gd, Th, Dy, Ho, Er, Tm, Yb, and Lv. Of these, Nd, Er, and Th arepreferred because they are commonly used in fluorescence applications.Attachment of a lanthanide atom (or a complex containing the lanthanideatom) to the oligonucleotide probe can be carried out usingsubstantially the same procedures used for tethering dyes thereto.Details on these procedures are described in, e.g., BioconjugateTechniques, Hermanson, ed. (Academic Press) (1996), which is herebyincorporated by reference in its entirety.

The quenching agent can be any agent that can be conjugated to a nucleicacid and preferably is characterized by an absorbance pattern that ismatched to cause complete or substantially complete quenching offluorescence emitted by the fluorescent label. The quenching agent canbe another fluorophore that absorbs emissions by the fluorescent labeland emits a different fluorescent emission pattern (i.e., during FRET)or the quenching agent can be formed of a material that absorbsfluorescent emissions by the fluorescent label but without acorresponding emission pattern. Examples of the former materials arethose described above with respect to the fluorescent label and whoseabsorption and emission patterns are well suited to achieve FRET.Examples of the latter materials include, without limitation, dyes, suchas 4-([4-(Dimethylamino)phenyl]azo)benzoic acid (dabcyl); and metalssuch as gold, silver, platinum, copper, cobalt, iron, iron-platinum,etc. Of these, the dye dabcyl and the metals gold, silver, and platinumare typically preferred.

The quenching agent can either be in the form of a small molecule suchas a dye, a particle such as a micro- or submicron-sized (i.e., nano-)particle, or in the form of a substrate that contains thereon asufficient density of the quenching agents such that the surface thereofis effectively a quenching surface. In one embodiment, the quenchingagent is a dye or a metal nanoparticle. In another embodiment, asubstrate have a quenching metal surface is utilized, such as asubstrate bearing a gold film thereon.

Assembly of the hairpin probe, e.g., on the metal surface, is carriedout in the presence of a spacing agent. Preferred spacing agents arenon-nucleic acid thiols. Exemplary spacing agents include, withoutlimitation, 3-mercapto-1-propanol, 1-mercapto-2-propanol,2-mercaptoethanol, 1-propanethiol, 1-butanethiol, 1-pentanethiol,3-mercapto-1,2-propanediol, 1-heptanethiol, 1-octanethiol, and1-nonanethiol. Ratios of non-nucleic acid thiol:DNA hairpin employed inthe assembly process are typically about 1:1 or greater, more preferablyabout 5:1 or greater. It is believed that the spacing agent providesspacing between individual molecules of DNA hairpin on the metalsurface. Chips assembled in the absence of spacing agent are, at best,poorly functional.

When multiple molecular beacons are used (e.g., in a microarray or othersimilar format) and each is conjugated to a fluorescent label, it ispreferable that the fluorescent labels can be distinguished from oneanother using appropriate detection equipment. That is, the fluorescentemissions of one fluorescent label should not overlap or interfere withthe fluorescent emissions of another fluorescent label being utilized.Likewise, the absorption spectra of any one fluorescent label should notoverlap with the emission spectra of another fluorescent label (whichmay result in undesired FRET that can mask emissions by the otherlabel).

The probes and molecular beacons identified in accordance with thepresent invention can be used in any of a variety of hybridization-basedapplications, typically though not exclusively detection procedures foridentifying the presence in a sample of a target nucleic acid molecule.By way of example, uses of the probes and molecular beacons aredescribed in greater detail in PCT Patent Application to Miller et al.,entitled “Hybridization-Based Biosensor Containing Hairpin Probes andUse Thereof,” filed Jan. 2, 2003, now WO 2004/061127, which is herebyincorporated by reference in its entirety.

EXAMPLES

The Examples set forth below are for illustrative purposes only and arenot intended to limit, in any way, the scope of the present invention.

Example 1 Hairpins Targeted to Bacillus anthracis pag Gene

A large sequence structure prediction from Bacillus anthracis is shownin FIG. 3 and depicts the “folding” of large sequences of DNA revealingseveral naturally occurring hairpins. The sequences are then isolatedfrom the full sequence and subjected to second structure prediction.FIGS. 4A-B show structural predictions for two of these excisedsequences.

These natural hairpins, BaPag668-706 (Pag 668) and BaPag1208-1241 (Pag1208), both appear to be good candidates for use as a molecular beacon,because each contains between about 30 to about 40 nucleotides and eachhas a E_(predict) between about −4 kcal/mol and about −12 kcal/mol.

Having confirmed that the selected hairpin(s) satisfy initial selectioncriteria, a final structural prediction of the sequence in duplex withits complement was computed (FIGS. 8A-B). This last prediction was doneprimarily to ensure that the hybridization of the two DNA sequences, andthus the disruption of the hairpin will be an energetically favorableprocess. Each of these duplexes have a predicted ΔΔG value that is aboutnine to ten-fold greater than the predicted E (ΔG) value for the hairpinalone, and therefore they are expected to favorably form a duplex withtheir targets.

The specificity of the hairpin of FIG. 4 for its target was supported bya BLAST search of the GenBank database using the BaPag 668-704 sequence.The results of this BLAST search are shown below in FIG. 5. Inparticular, the BLAST results indicate that only sequences from Bacillusanthracis, the target organism, have high scores; whereas other“matching sequences from non-target organisms have significantly lowerscores. In this instance, a clear demarcation exists between targetscores (of 78) and non-target scores (of 42 and lower). Thisdemonstrates that this hairpin will be specific for its target.

Example 2 Hairpins Targeted to Staphylococcus aureus Genome

Two DNA hairpins, AH2 and BH2, were designed to incorporate portions ofthe Staphylococcus aureus genome (Genbank Accession AP003131, which ishereby incorporated by reference in its entirety). The AH2 sequenceappears to target an intergenic region between ORFID:SA0529 andORFID:SA0530, and the BH2 sequence appears to target an intergenicregion between ORFID:SA0529 and ORFID:SA0530 but also includes severalbases within the latter open reading frame.

A segment of the complete Staphylococcus aureus genome was obtained fromthe GenBank database and the secondary structure of the obtained segmentwas predicted using computer program RNAStructure version 3.7 (Mathewset al., J. Mol. Biol. 288:911-940 (1999), which is hereby incorporatedby reference in its entirety), as shown in FIG. 6. From this predictedstructure, two naturally occurring hairpins were identified, onedesignated AH2 and the other designated BH2 (FIG. 6).

Having identified these two sequences, these sequences were isolatedfrom the larger sequence and subjected to a second structure predictionas described above. The predicted structure of AH2 is characterized by apredicted free energy value of about −6.1 kcal/mol (FIG. 7A) and thepredicted structure of BH2 is characterized by a predicted free energyvalue of about −3.5 kcal/mol (FIG. 7B). Both are within the size rangeof about 30-40 nucleotides.

Having selected AH2 and BH2, a final structural prediction of theduplexes (AH2 and BH2 with their respective complements) was carried outto determine their ΔΔG value. The duplex containing AH2 was predicted tohave a free energy value of −32.2 kcal/mol and the duplex containing BH2was predicted to have a free energy value of −35.5 kcal/mol (FIGS.8C-D). These values indicate that the hybridization between the hairpinand its target will be an energetically favorable process. A BLASTsearch was independently performed using the AH2 and BH2 sequences, theresults indicating that only segments of the Staphylococcus aureusgenome contain highly related nucleotide sequences.

Example 3 Hairpins Targeted to Other Pathogen

This process described above and exemplified in Examples 1-2 has alsobeen performed using Exophiala dermatitidis 18S ribosomal RNA genesequences to identify hairpin probes that can be used to identify thetarget gene (and organism); Trichophyton tonsurans strain 18S ribosomalRNA gene sequences to identify hairpin probes that can be used toidentify the target gene (and organism); and Bacillus cereus genomic DNAto identify hairpin probes that can be used to identify the target DNA(and organism). These sequences have been reported in PCT PatentApplication to Miller et al., entitled “Hybridization-Based BiosensorContaining Hairpin Probes and Use Thereof,” filed Jan. 2, 2003, now WO2004/061127, which is hereby incorporated by reference in its entirety.

Example 4 Hairpins Favorably Hybridize with their Target DNA

Samples of BaPag668 and BaPag1208, both alone and mixed with equalamount of complement, were run on a native polyacrylamide gel. Thepurpose of this experiment was two-fold: (1) to demonstrate that, aspredicted, the designed hairpins form only one preferred structure, and(2) to provide another example that the hairpins will favorablyhybridize with their target DNA. The results are shown in FIG. 9.

The presence of single bands in Lanes 1 and 3 is evidence that thehairpins preferentially adopt one structure. This claim can be madeconfidently because the distance non-duplexed DNA migrates through apolyacrylamide gel is based on both the size (molecular weight) andshape of the molecule in question. Any variations from the predictedstructure would either enhance or retard the variant's migration throughthe gel, thus creating multiple bands.

The upward shift seen in Lanes 2 and 4 is indicative of the addition ofmass that occurs during the hybridization of the hairpins with theirtargets. The increased contrast of the bands in Lanes 2 and 4 also givesindication that the hairpins are successfully forming double-strandedduplexes with their targets, as the dye used preferentially bindsdouble-stranded regions of DNA.

Example 5 Thermal Melting Curves for DNA Hairpin Probes

Determination of the presence of an ordered secondary structure wasaccomplished via the procurement of thermal melting profiles. Allmelting temperatures of BaPag668-706, BaPag1208-1241, AH2, and BH2 werefound to be concentration independent. As discussed by others (Inglesbyet al., “Anthrax as a Biological Weapon: Medical and Public HealthManagement,” J. Am. Med. Assoc. 281:1735-1745 (1999), which is herebyincorporated by reference in its entirety), the observed concentrationindependence is a strong indicator of the presence of an orderedsecondary structure, presumed to be the desired hairpins. The unmodifiedhairpins were then mixed with a ten-fold excess of complementary DNA anda second series of melting profiles were obtained (FIGS. 10E-H). As wasexpected, introduction of complement to the hairpins produced a biphasictransition curve, with the first transition corresponding to thelinearization of the target DNA, which is also believed to possessordered secondary structure, and the second, higher temperature,transition corresponding to the melting point of the duplex DNA.

Example 6 Solution-Phase Performance of Beacon BaPag668-706

To provide an initial indication of the ability of the BaPag668-706probe to function as a molecular beacon, the response to target DNA ofBaPag668-706 when modified with a 5′-fluorescein and a 3′-dabcyl. Themodified BaPag668-706 beacon was mixed with increasing concentrations oftarget DNA in aluminum foil covered eppendorf tubes. After approximatelyone hour at room temperature, fluorescence measurements were procured todetermine the efficacy of the beacon. As shown in FIG. 11, BaPag668-706exhibits minimal fluorescence alone, and, as expected, addition of thetarget complementary oligonucleotide causes fluorescence to increase ina concentration-dependent manner.

Example 7 Performance of BaPag1208 Immobilized on an Au-Film

The performance of the functionalized hairpins as Au-immobilized DNAsensors was examined. BaPag1208 was immobilized onto an Au film in muchthe same manner as has previously been reported (Du et al.,“Hybridization-based Unquenching of DNA Hairpins on Au Surfaces:Prototypical “Molecular Beacon” Biosensors,” J. Am. Chem. Soc.125:4012:4013 (2003), which is hereby incorporated by reference in itsentirety), with the only major change being the use of 0.5 M NaCl bufferas the diluent as opposed to deionized water. BaPag1208 was initiallyimmobilized in a 1:10 ratio with mercapto propanol, the results of whichare shown in FIGS. 12A-C.

When immobilized on an Au-film in a 1:10 ratio with mercaptopropanol,BaPag1208 shows greater than an 18-fold increase (FIGS. 12A-C) influorescence intensity in response to incubation in a 2.5 μM targetsolution. When the concentration of the target solution is lowered to1.0 μM, the observed response drops to about 10-fold, which is stillsignificant (FIGS. 12D-F).

Despite reports that 1:10 ratio of beacon to mercaptopropanol providedfor the best signal to noise ratio for more traditional beacons,additional studies suggested that for BaPag1208, a 1:1 ratio may providea more effective beacon. As such, BaPag1208 was immobilized onto anAu-film with mercaptopropanol in a 1:1 ratio and subjected to the sametarget concentrations as described previously. As can be clearly seen inFIGS. 13A-C, when immobilized in a 1:1 ratio with mercaptopropanol,BaPag1208 shows a superior response to target over that observed whenthe immobilization ratio is 1:10. This increased response is especiallysignificant at lower concentrations as is evidenced by the greater than20-fold intensity increase observed after incubation in 1.0 μM target.(FIG. 13D-F).

Initial studies as to the sensitivity of BaPag1208-1241 when immobilizedonto an Au-film have been started and are summarized in FIG. 14.BaPag1208-1241 immobilized beacons have shown a nearly a 10-foldresponse to a 1.0 mL solution of 1.0 nM target (1.0 pmol). Studiesplanned for the very near future should elucidate the absolute limit ofdetection for a solution of synthetic and native targets.

Example 8 Performance of AH2 and BH2 Immobilized on an Au-Film

To examine the suitability of the “partial gene folding” derived beaconsin such a scheme, the Staphylococcus aureus probes AH2 and BH2 wereobtained modified with a 5′-thiol (allowing for attachment to Au filmusing standard chemistry), and either a 3′-rhodamine (AH2) or a 3′-Cy5(BH2). These two probes were concurrently assembled in a 1:1 ratio inthe presence of mercaptopropanol on two Au films. Individual films werethen treated with solutions of either AH2-complement or BH2-complement.Addition of 1.0 μM AH2-complement yielded a chip with significantfluorescence around 585 nm (FIG. 15C), while addition of 1.0 μMBH2-complement produced weak, but still observable Cy5 fluorescence (675nm) (FIG. 15F). A partial reason for the weak signal observed from theCy5 is due to the small absorption cross section for Cy5 in greenwavelengths. Indeed, using an AH2-Cy5 functionalized surface, excitationat 633 nm (cross section 8 times greater than at 514 nm) produced twiceas much fluorescence intensity from Cy5. These results suggest thatalthough differentiating multiple targets with only a single lightsource is not yet optimized, co-immobilization of two probes produces afunctional chip.

Example 9 Calculated Hybridization Energies for Folding-Derived andModified Beacons

It is difficult to rigorously compare folding-derived to modifiedbeacons, since changing the sequence obviously alters more than oneexperimental parameter. However, the effects of modification can bepredicted, as shown by the calculations in FIGS. 16 and 17. The terminiof probes BaPag668-706 and BaPag1208-1241 were first extended by theself-complementary sequence [d(CGACG)]₂, then the hybridization energycalculated (FIGS. 16B and 17B, respectively). Second, five bases wereremoved from each end of BaPag668-706 and BaPag1208-1241, replaced with[d(CGACG)]₂, and the hybridization energy again calculated (FIGS. 16Cand 17C, respectively). In each case, calculated ΔΔG values were lessfavorable for the modified beacons than for the probes derived directlyfrom folding.

The fact that the hybridization product of the new beacon isenergetically superior to that of the traditional design should lead thenew beacon to have a higher sensitivity. The binding free energy forhybridization ΔG_(bind) is related to the observed equilibriumassociation constant K_(A) by: −ΔG_(bind)=−RTlnK_(A), where T is thetemperature and R the universal gas constant (Riccelli et al.,“Hybridization of Single-stranded DNA Targets to ImmobilizedComplementary DNA Probes: Comparison of Hairpin Versus Linear CaptureProbes,” Nucl. Acids Res. 29: 996-1004 (2001), which is herebyincorporated by reference in its entirety). The use of hairpins thathave 100% sequence participation in duplex formation allows for a moreenergetically favorable duplex than would exist for a hairpin thatcontains non-specific termini. Thus, the duplex that forms the moreenergetically favorable dimer will be expected to bind much moretightly, and therefore is expected to be more sensitive. Highlysensitive detection schemes are preferred for rapid detection andidentification of pathogens in a clinical sample.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A method of identifying a hairpin nucleic acid probe that hybridizesover its entire length to a target nucleic acid molecule, the methodcomprising: providing a target nucleic acid sequence that is larger thanabout 100 nucleotides in length; predicting a folded structure of thetarget nucleic acid sequence; identifying a nucleotide sequence of ahairpin within the folded structure of the target nucleic acid sequence;and predicting a folded structure for the identified nucleotide sequenceof the hairpin, in the absence of other nucleotides of the targetnucleic acid sequence, wherein the folded structure of a hairpin thathas a predicted E value of at most about −3 kcal/mol is a probe thathybridizes over its entire length to the target nucleic acid molecule.2. The method according to claim 1 wherein the nucleotide sequence ofthe hairpin is between about 12 and about 60 nucleotides in length. 3.The method according to claim 1 wherein the folded structure of thehairpin has a predicted E value of between about −4 kcal/mol and about−12 kcal/mol.
 4. The method according to claim 1 further comprising:predicting a folded structure of a duplex formed between the hairpin andits complement.
 5. The method according to claim 4 further comprising:determining whether duplex formation is energetically favorable.
 6. Themethod according to claim 1 further comprising: performing a databasesearch for nucleotide sequences that are similar to the identifiednucleotide sequence of the hairpin.
 7. The method according to claim 6further comprising: determining, from the results of the performeddatabase search, whether a clear demarcation exists between scores fortarget nucleic acid sequences and scores for non-target nucleic acidsequences.
 8. A method of preparing a molecular beacon comprising:providing a hairpin nucleic acid probe identified according to themethod of claim 1; and tethering a fluorescent label and a quenchingagent to the opposed termini of the provided hairpin nucleic acid probeto form a molecular beacon, wherein the molecular beacon issubstantially non-fluorescent in the absence of a nucleic acidcomplementary to the hairpin nucleic acid probe.
 9. The method accordingto claim 8, wherein said providing comprises: synthesizing a nucleicacid molecule corresponding to the nucleotide sequence of the hairpinprobe.
 10. The method according to claim 8, wherein the fluorescentlabel is tethered to the 5′ terminus and the quenching agent is tetheredto the 3′ terminus.
 11. The method according to claim 8, wherein thefluorescent label is tethered to the 3′ terminus and the quenching agentis tethered to the 5′ terminus.
 12. The method according to claim 8,wherein the quenching agent is a solid surface.
 13. The method accordingto claim 8, wherein the quenching agent is a micro- or nano-particle.14. The method according to claim 8, wherein the fluorescent label is afluorescent dye, semiconductor quantum dot, lanthanide atom-containingcomplex, or fluorescent protein.
 15. The method according to claim 8,wherein the quenching agent is a metal or4-([4-(Dimethylamino)phenyl]azo)benzoic acid.
 16. The method accordingto claim 15, wherein the metal is gold, silver, platinum, copper,cobalt, iron, or iron-platinum.
 17. A method of preparing a hairpinnucleic acid molecule comprising: synthesizing a hairpin nucleic acidmolecule identified according to the method of claim
 1. 18. A method ofidentifying a hairpin nucleic acid probe that hybridizes over its entirelength to a target nucleic acid molecule, the method comprising:providing a target nucleic acid sequence that is larger than about 100nucleotides in length; predicting a folded structure of the targetnucleic acid sequence; identifying a nucleotide sequence of a hairpinwithin the folded structure of the target nucleic acid sequence, thehairpin being between about 12 and about 60 nucleotides in length; anddetermining whether (i) self-folding of the identified hairpin and (ii)hairpin binding over its entire length to the target nucleic acidmolecule will be energetically favorable.